Abstract
Inorganic phosphate (Pi) is an essential component for structure and metabolism. PiT1 (SLC20A1) and PiT2 (SLC20A2) are members of the mammalian type-III inorganic phosphate transporters. SLC20A2 missense variants are associated with primary brain calcification. MicroRNAs (miRNAs) are endogenous noncoding regulatory RNAs, which play important roles in post-transcriptional gene regulation. MicroRNA-9 (miR-9) acts at different stages of neurogenesis, is deeply rooted in gene networks controlling the regulation of neural progenitor proliferation, and is also linked with cancers outside the nervous system. We evaluated possible interactions between miR-9 and the phosphate transporters (PiT1 and PiT2). SLC20A2, platelet-derived growth factor receptor beta (PDGFRB) and Fibrillin-2 (FBN2) showed binding sites with high affinity for mir-9, in silico. miR-9 mimic was transfected into HEK293 cells and expression confirmed by RT-qPCR. Overexpression of miR-9 in these cells caused a significant reduction in PiT2 and FBN2. PDGFRB appeared to be decreased, but was not significantly down-regulated in our hands. PiT1 showed no significant difference relative to controls. The down-regulation of PiT2 protein by miR-9 was confirmed by western blotting. In conclusion, we showed miR-9 can down-regulate PiT2, in HEK293 cells.
Similar content being viewed by others
References
Alexiou P, Maragkakis M, Papadopoulos GL et al (2009) Lost in translation: an assessment and perspective for computational microrna target identification. Bioinformatics 25:3049–3055. doi:10.1093/bioinformatics/btp565
Bøttger P, Pedersen L (2002) Two highly conserved glutamate residues critical for type III sodium-dependent phosphate transport revealed by uncoupling transport function from retroviral receptor function. J Biol Chem 277:42741–42747. doi:10.1074/jbc.M207096200
Bøttger P, Pedersen L (2011) Mapping of the minimal inorganic phosphate transporting unit of human PiT2 suggests a structure universal to PiT-related proteins from all kingdoms of life. BMC Biochem 12:21. doi:10.1186/1471-2091-12-21
Bøttger P, Hede SE, Grunnet M et al (2006) Characterization of transport mechanisms and determinants critical for Na + −dependent Pi symport of the PiT family paralogs human PiT1 and PiT2. Am J Physiol Cell Physiol 291:C1377–C1387. doi:10.1152/ajpcell.00015.2006
Burney RO, Hamilton AE, Aghajanova L et al (2009) MicroRNA expression profiling of eutopic secretory endometrium in women with versus without endometriosis. Mol Hum Reprod 15:625–631. doi:10.1093/molehr/gap068
Cao D-D, Li L, Chan W-Y (2016) MicroRNAs: key regulators in the central nervous system and their implication in neurological diseases. Int J Mol Sci 17:842. doi:10.3390/IJMS17060842
Chen X, Zhu L, Ma Z et al (2015) Oncogenic miR-9 is a target of erlotinib in NSCLCs. Sci Rep 5:17031. doi:10.1038/srep17031
Coolen M, Katz S, Bally-Cuif L (2013) miR-9: a versatile regulator of neurogenesis. Front Cell Neurosci 7:220. doi:10.3389/fncel.2013.00220
Giusti SA, Vogl AM, Brockmann MM et al (2014) MicroRNA-9 controls dendritic development by targeting REST. 1–22. doi:10.7554/eLife.02755
Inden M, Iriyama M, Takagi M et al (2013) Localization of type-III sodium-dependent phosphate transporter 2 in the mouse brain. Brain Res 1531:75–83. doi:10.1016/j.brainres.2013.07.038
Jensen N, Schrøder HD, Hejbøl EK et al (2013) Loss of function of Slc20a2 associated with familial idiopathic basal ganglia calcification in humans causes brain calcifications in mice. J Mol Neurosci 51:994–9. doi:10.1007/s12031-013-0085-6
Jensen N, Autzen JK, Pedersen L (2016) Slc20a2 is critical for maintaining a physiologic inorganic phosphate level in cerebrospinal fluid. Neurogenetics 17:125–130. doi:10.1007/s10048-015-0469-6
Kavanaugh MP, Miller DG, Zhang W et al (1994) Cell-surface receptors for gibbon ape leukemia virus and amphotropic murine retrovirus are inducible sodium-dependent phosphate symporters. Proc Natl Acad Sci 91:7071–7075. doi:10.1073/pnas.91.15.7071
Keasey MP, Kang SS, Lovins C, Hagg T (2013) Inhibition of a novel specific neuroglial integrin signaling pathway increases STAT3-mediated CNTF expression. Cell Commun Signal 11:35. doi:10.1186/1478-811X-11-35
Keasey MP, Lemos RR, Hagg T, Oliveira JRM (2016) Vitamin-D receptor agonist calcitriol reduces calcification in vitro through selective upregulation of SLC20A2 but not SLC20A1 or XPR1. Sci Rep 6:25802. doi:10.1038/srep25802
Keller A, Westenberger A, Sobrido MJ et al (2013) Mutations in the gene encoding PDGF-B cause brain calcifications in humans and mice. Nat Genet 45:1077–82. doi:10.1038/ng.2723
Legati A, Giovannini D, Nicolas G et al (2015) Mutations in XPR1 cause primary familial brain calcification associated with altered phosphate export. Nat Genet 47:579–81. doi:10.1038/ng.3289
Lemos RR, Ramos EM, Legati A et al (2015) Update and mutational analysis of SLC20A2: a major cause of primary familial brain calcification. Hum Mutat 36:489–495. doi:10.1002/humu.22778
Liu W, Zhao N, Li X, et al (2015) A novel FBN2 mutation in a Chinese family with congenital contractural arachnodactyly. 5:163–166
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and. Methods 25:402–408. doi:10.1006/meth.2001.1262
Mccartney E, Squier W (2014) Patterns and pathways of calcification in the developing brain. 1009–1015. doi:10.1111/dmcn.12493
Nicolas G, Pottier C, Maltête D et al (2013) Mutation of the PDGFRB gene as a cause of idiopathic basal ganglia calcification. Neurology 80:181–187. doi:10.1212/WNL.0b013e31827ccf34
Nisancioglu MH, Wallgard E, Niaudet C et al (2010) Pericytes regulate the blood–brain barrier. 6–11. doi:10.1038/nature09522
Press D (2016) Prognostic role of miR-9 expression in various human malignant neoplasms: a meta-analysis. 3039–3047
Qi XJ, Wang JF, Wang GD et al (2016) Pivotal role of microRNA-9 in osteosarcoma tumorigenesis and tumor progression. Genet Mol Res 15:1–10. doi:10.4238/gmr.15017318
Sapio L, Naviglio S (2015) Inorganic phosphate in the development and treatment of cancer: a Janus Bifrons? World J Clin Oncol 6:198–201. doi:10.5306/wjco.v6.i6.198
Shin VY, Chu KM (2014) MiRNA as potential biomarkers and therapeutic targets for gastric cancer. World J Gastroenterol 20:10432–10439. doi:10.3748/wjg.v20.i30.10432
Stern-Ginossar N, Elefant N, Zimmermann A et al (2007) Host immune system gene targeting by a viral miRNA. Science 317:376–81. doi:10.1126/science.1140956
Uckert W, Willimsky G, Pedersen FS et al (1998) RNA levels of human retovirus receptor Pit1 and Pit2 do not correlate with infectibility by three retroviral vector pseudotypes. Hum Gene Ther 2627:2619–2627
Waldman SA, Terzic A (2008) MicroRNA signatures as diagnostic and therapeutic targets. Clin Chem 54:943–944. doi:10.1373/clinchem.2008.105353
Wallingford MC, Chia JJ, Leaf EM et al (2016) SLC20A2 deficiency in mice leads to elevated phosphate levels in cerbrospinal fluid and glymphatic pathway-associated arteriolar calcification, and recapitulates human idiopathic basal ganglia calcification. Brain Pathol 3:64–76. doi:10.1111/bpa.12362
Wang C, Li Y, Shi L et al (2012) Mutations in SLC20A2 link familial idiopathic basal ganglia calcification with phosphate homeostasis. Nat Genet 44:254–6. doi:10.1038/ng.1077
Zhang J, Chintalgattu V, Shih T et al (2011) MicroRNA-9 is an activation-induced regulator of PDGFR-beta expression in cardiomyocytes. J Mol Cell Cardiol 51:409–418. doi:10.1016/j.yjmcc.2011.06.001
Zhang H, Shykind B, Sun T (2012) Approaches to manipulating micrornas in neurogenesis. Front Neurosci 6:1–13. doi:10.3389/fnins.2012.00196
Acknowledgments
This work was funded by grants originally from CNPq and FACEPE.
Author information
Authors and Affiliations
Corresponding authors
Additional information
An erratum to this article is available at http://dx.doi.org/10.1007/s12031-017-0913-1.
Rights and permissions
About this article
Cite this article
Paiva, D., Keasey, M. & Oliveira, J.R.M. MiR-9-5p Down-Regulates PiT2, but not PiT1 in Human Embryonic Kidney 293 Cells. J Mol Neurosci 62, 28–33 (2017). https://doi.org/10.1007/s12031-017-0906-0
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12031-017-0906-0